Fig. 4: Single-molecule kinetic analysis of the nNEs.
From: A rhythmically pulsing leaf-spring DNA-origami nanoengine that drives a passive follower
a, Schematic of the smFRET assay. FRET between the Cy3 (cyan) donor and the Cy5 (magenta) acceptor dyes monitors the distance between the two arms of the nNEs. b, Representative FRET time traces of single nNEs under varying conditions. Static traces are observed in the absence of either HT–T7RNAP or NTPs; dynamic traces are observed only when both were present. Arrows, dye photobleaching (PB). Right: histograms for each trace showing the low- (blue) and high-FRET (red) states. c, Representative fluorescence time trajectory of a single nNE in the presence of 5 mM of each NTP. The anticorrelated intensities of Cy3 and Cy5 are monitored until Cy5 and/or Cy3 photobleach. The smFRET trajectory (black) shows multiple transitions between two dominant FRET states. d, Representative segmentation analysis of a dynamic smFRET trace reveals the cycle time (τcycle) of nNE opening–closing events, subdivided into low-FRET-state time (τO), transition time from low- to high-FRET (τt-C), high-FRET-state time (τC), and transition time from high- to low-FRET (τt-O). Green arrows, abortive transcription events. e,f, Cumulative distributions for τcycle (e) and individual time components (τO, τt-C, τC, τt-O) (f) for 1 mM and 5 mM NTP conditions (lighter and darker colour, respectively). Number of molecules (N) and transitions (n) are shown at the bottom of each plot. τO and τC were fitted with double-exponential functions, τtF and τtR were fitted with gamma functions to obtain their respective transition time constants. Errors represent s.d. of three biological replicates. g, Single-molecule traces from non-equilibrium ‘NTP-switch’ experiments with segments before addition of NTPs (−NTP, static), after addition of NTPs (+NTPs, dynamic) and after removal of NTP (−NTP, halted). Grey axis break indicates the dark period between segments during which the buffer was exchanged. h, Heat map of all molecules in the respective states, representing a cumulative behaviour. Static molecules were observed in the low-FRET state in −NTP static phase. Upon NTP addition, the same molecules exhibited dynamic behaviour with transition between low-FRET (L) and high-FRET (H) states. When NTPs were washed out, molecules remain either in the H or L state. Colour bar, number of molecules in a particular state in the heat map.
